Adiponectin promoter and use thereof

ABSTRACT

The present invention relates to a DNA having a promoter region containing regulatory sequences of human adiponectin gene, transformants transformed with the DNA, a screening method of a compound which can enhance the human adiponectin promoter activity, which the transformants are used, and the screening kit, and a screening method of a preventive/therapeutic medicine for syndromes such as Syndrome X, metabolic syndrome, multiple risk factor syndrome, insulin resistant syndrome, deadly quartet, and visceral fat syndrome, metabolic disorders such as diabetes, obesity, hypercholesterolemia, and hyperlipoproteinemias, hyperlipidemia, arteriosclerosis, hypertonia, circulatory system disease, and hyperphagia and a pharmaceutical composition obtained by using them.

TECHNICAL FIELD

The present invention relates to a promoter containing novel regulatorynucleotide sequences for gene expression and use thereof. Concretely, itrelates to a DNA containing the promoter region with regulatorysequences of human adiponectin gene, transformants transformed with theDNA, and screening methods of compounds or salts thereof that accelerateadiponectin promoter activity via the regulatory sequences.

BACKGROUND ART

Recently, the adipose tissue has been shown to be an endocrine organthat actively produces and secretes many bioactive substances, calledadipocytokines, which play a crucial role in the control of systemicglucose and lipid metabolism (Nature, 414:799-806, 2001). Adiponectin isone of adipocytokines identified as the most abundantly-expressing genein human adipocytes and as a hormone (Biochemical and BiophysicalResearch Communication, 221:286-289, 1996). Adiponectin is specificallyproduced and secreted in adipocytes, and exists abundantly in the blood.

Many research results have shown that adiponectin is an antidiabetic,antiarteriosclerotic, and antiobestic hormone, which is closelyassociated with the onset and progress of metabolic diseases. Forexample, plasma adiponectin concentration was decreased in patients withischemic heart diseases or insulin-resistant diabetes (Circulation,100:2473-2476, 1999; Arteriosclerosis, Thrombosis, and Vascular Biology,20:1595-1599, 2000). Furthermore, in patients with mutations in theadiponectin gene, the decrease in plasma adiponectin concentration wasassociated with the onset of insulin-resistant diabetes oratherosclerosis (Diabetes, 50: 1126-1133, 2002). In obese diabeticmonkeys, the progressive reduction in plasma adiponectin was deeplyassociated with the aggravation of insulin resistance (Diabetes,50:1126-1133, 2001). In adiponectin-deficient mice, diabetes was causedby the load of high sucrose and high fat diet, and neointimal thickeningafter artery injury was remarkably accentuated. Adenovirus-mediatedsupplement of adiponectin into the knockout mice eminently improved thelesion (Nature Medicine, 8:731-737, 2002, Journal of BiologicalChemistry, 277:37487-37491, 2002). Moreover, adenoviral supplement ofadiponectin into apolipoprotein E-deficient mice suppressed the progressof arteriosclerosis (Circulation, 106:2767-2770, 2002). When recombinantadiponectin protein was administrated in metabolically-impaired mice,the improvement of insulin resistance and hypoglycemic action were seen(Nature Medicine, 7:941-946, 2001, Nature Medicine, 7:947-953, 2001).According to these facts, the therapy raising plasma adiponectinconcentration is thought to be effective in improving the condition ofpatients with hypoadiponectinemia. However, plasma adiponectinconcentration in human, which is 5-20 μg/ml (Biochemical and BiophysicalResearch Communication, 257:79-83, 1999), is extremely high comparedwith other blood hormones. Considering this respect, in patients withhypoadiponectinemia, it is thought that the substitution therapy ofrecombination adiponectin protein to normalize plasma concentrationwould be accompanied by various great difficulties in continuoushigh-dose administration of the protein or the prevention of in vivoenzymatic hydrolysis of the protein.

DISCLOSURE OF THE INVENTION

Though the production of proteins can be controlled at various stages invivo, the transcriptional regulation is the most fundamental step. Thus,modifying the level of disease-associated protein at the transcriptionalstage is one of the powerful means to treat diseases. Gene transcriptionis regulated via the promoter and enhancer region located inneighborhood of the nucleotide sequence transcribed into messenger RNA.For transcriptional regulation, a kind of proteins, calledtranscriptional regulatory factors, is needed to bind to the nucleotidesequences, called the regulatory sequences, in the promoter and enhancerregions of the gene. Among those transcriptional regulatory factors, agroup of proteins called nuclear receptors have a unique character thattheir activities can be regulated by interacting with small moleculescalled ligands. For example, PPARγ (peroxisome proliferator-activatedreceptor γ) is an important nuclear receptor for adiposedifferentiation. PPARγ forms a heterodimer with another nuclear receptorRXR (retinoid X receptor) and specifically binds to the regulatorysequence called PPRE (peroxisome proliferator-activated receptorresponsive element) in the promoter or enhancer region of the genes.PPARγ activity is regulated by endogenous unknown ligands or exogenousligands such as thiazolidinedione derivatives (Annu. Rev. Biochem.,70:341-367, 2001). The regulatory sequence PPRE is comprised of thecharacteristic nucleotide sequence that is represented by5′-AGGTCAnAGGTCA-3′ (SEQ ID NO:15), which is different from every kindof genes with PPRE. When creating the medicine that can exhibit efficacyby changing the adiponectin production at the transcriptional stage,identification of the regulatory sequence in the promoter or enhancerregion of the adiponectin gene is quite useful in constructing theefficient and best screening system for discovering potential compounds.Transformants can be created by connecting a DNA containing theidentified regulatory sequences to a suitable reporter gene, andtransforming host cells with the DNA. The transformants can be used as auseful screening system of preventive and/or therapeutic medicines formetabolic disorder such as diabetes, obesity, hypercholesterolemia, andhyperlipoproteinemias, etc., hyperlipidemia, arteriosclerosis,hypertonia, circulatory system disease, and hyperphagia, etc., which canact through the induction of adiponectin gene expression. Moreover, thetransformants can be also used as the useful screening system ofpreventive and/or therapeutic medicines for various syndromes (SyndromeX, metabolic syndrome, multiple risk factor syndrome, insulin resistancesyndrome, deadly quartet, visceral fat syndrome, etc.) caused by theabove diseases. However, regulatory sequences related to the control ofhuman adiponectin gene expression has not been identified so far and nomethod can substantially and effectively screen any accelerators of thehuman adiponectin gene expression.

As a result of the present inventors' repeated researches to establishthe screening method that can explore accelerators of the adiponectingene expression, they discovered and identified the characteristicregulatory sequence PPRE and LRH-RE (liver receptor homologue-1responsive element) of adiponectin gene in the 5′ upstream promoterregion of human adiponectin gene. Moreover, they found that PPRE andLRH-RE modulate the physiological human adiponectin promoter activity.According to these findings, the present inventors have completed thisinvention as a result of more researches.

That is, the present invention relates to the followings:

-   (1) A DNA comprising a promoter region having the nucleotide    sequence presented by SEQ ID NO:1 which comprises a regulatory    sequence of a human adiponectin gene.-   (2) The DNA according to the above (1), which consists of a promoter    region having the nucleotide sequence presented by SEQ ID NO:1 which    comprises a regulatory sequence of a human adiponectin gene.-   (3) The DNA according to the above (2), wherein the regulatory    sequence is a sequence containing PPRE (Peroxisome    Proliferator-activated Receptor Responsive Element).-   (4) The DNA according to the above (2), wherein the regulatory    sequence is a sequence containing LRH-RE (Liver Receptor Homologue-1    Responsive Element).-   (5) The DNA according to the above (2), wherein the regulatory    sequence is the nucleotide sequence presented by SEQ ID NO:2.-   (6) he DNA according to the above (2), wherein the regulatory    sequence is the nucleotide sequence presented by SEQ ID NO:3.-   (7) The DNA according to the above (2), wherein the regulatory    sequence is a nucleotide sequence comprising the nucleotide sequence    presented by SEQ ID NO:2 and the nucleotide sequence presented by    SEQ ID NO:3.-   (8) The DNA according to the above (2), which the regulatory    sequence is the nucleotide sequence presented by SEQ ID NO:4.-   (9) A recombinant plasmid DNA comprising the DNA according to the    above (2).-   (10) The recombinant plasmid DNA according to the above (9), which    is capable of expressing a structural gene under control of the    promoter region comprising the regulatory sequence of human    adiponectin gene can express.-   (11) A transformant transformed with the recombinant plasmid DNA    according to the above (9) or (10).-   (12) A screening method of a compound which is capable of enhancing    human adiponectin promoter activity or a salt thereof, which    comprising using the transformant according to the above (11).-   (13) A screening method of a preventive and/or therapeutic medicine    for syndromes selected from syndrome X, metabolic syndrome, multiple    risk factor syndrome, insulin resistance syndrome, deadly quartet,    and visceral fat syndrome, which comprises using the transformant    according to the above (11).-   (14) The screening method according to the above (13), which a    disorder as a etiology of the syndrome is diabetes, obesity,    hypercholesterolemia, hyperlipoproteinemias, hyperlipidemia,    arteriosclerosis, hypertonics, circulatory system disease, or    polyphagies.-   (15) A screening kit of a compound which is capable of enhancing    human adiponectin promoter activity or a salt thereof, which    comprises using the transformant according to the above (11).-   (16) A screening kit of a preventive and/or therapeutic medicine for    syndrome selected from syndrome X, metabolic syndrome, multiple risk    factor syndrome, insulin resistance syndrome, deadly quartet, and    visceral fat syndrome, which comprises using the transformant    according to the above (11).-   (17) A compound which is capable of enhancing human adiponectin    promoter activity or a salt thereof, which is obtainable by using    the screening method according to the above (12).-   (18) A preventive and/or therapeutic medicine for syndrome selected    from syndrome X, metabolic syndrome, multiple risk factor syndrome,    insulin resistance syndrome, deadly quartet, and visceral fat    syndrome, which is obtainable by using the screening method    according to the above (13).-   (19) A compound which is capable of enhancing human adiponectin    promoter activity or a salt thereof, which is obtainable by using    the screening kit according to the above (15).-   (20) A preventive and/or therapeutic medicine for syndromes selected    from syndrome X, metabolic syndrome, multiple risk factor syndrome,    insulin resistance syndrome, deadly quartet, and visceral fat    syndrome, which is obtainable by using the screening kit according    to the above (16).-   (21) A pharmaceutical composition which comprises the compound which    is capable of enhancing human adiponectin promoter activity    according to the above (17) or (19) or a salt thereof.-   (22) A pharmaceutical composition which comprises the preventive    and/or therapeutic medicine for syndromes selected from Syndrome X,    metabolic syndrome, multiple risk factor syndrome, insulin    resistance syndrome, deadly quartet, and visceral fat syndrome    according to the above (18) or (20).

The DNA of the present invention containing the regulatory sequence PPREor LRH-RE in the promoter region of human adiponectin gene or containingboth PPRE and LRH-RE may be anything as long as it contains theregulatory sequence and has the adiponectin promoter activity.Concretely, it may be anything as long as it contains the nucleotidesequence presented by SEQ ID NO:1, the complimentary sequence, or theseparts. Moreover, it may be anything of genomic DNA, cDNA, and asynthetic DNA derived from human.

Concretely, the DNA that contains the regulatory sequence PPRE and/orLRH-RE in the promoter region of human adiponectin gene of the presentinvention can be obtained as follows.

Firstly, using primers corresponding to the nucleotide sequence of thepromoter region of human adiponectin gene that has already been reported(Int. J. Obes. Relat. Metab. Disord., 24:861-868, 2000), target DNAfragments are amplified by PCR method using genomic DNA (for example,clontech) derived from human tissues as template. The obtained DNA maybe cloned into plasmid and the nucleotide sequence may be determined.According to the purpose, the obtained DNA may be used as it is, beingdigested by restriction enzymes, or being ligated to linkers. Moreover,a method of connecting a transcriptionally detectable reporter gene tothe downstream of the obtained DNA is preferred as a method to examinethe promoter activity. Though luciferase gene, chloramphenicol acetyltransferase gene, alkaline phosphatase gene, and beta-galactosidasegene, etc. are used widely as a reporter gene, even any other structuralgenes are possible to use it if the gene product can be detected.

As a host for transformation with the above recombinant plasmid DNA,yeasts, insect cells, and animal cells, etc. can be used. For example,yeasts include Saccharomyces cerevisiae AH22R⁻, NA87-11A, and DKD-5D,etc. As insect cells, for example, mamestra brassicoe Sf9 cells andsilkworm BmN cells, etc. can be used. As animal cells, for example,monkey COS-1 cells, COS-7 cells, Chinese hamster CHO cells, mouse Lcells, 293T cells, 3T3-L1 cells, human HEK293 cells, HepG2 cells, albedoadipocytes, and differentiation-induced cells by suitabledifferentiation condition, etc. can be used.

Yeasts are transformed by a properly modified method that is describedin, for example, Proc. Natl. Acad Sci. USA, vol. 75, 1929 (1978). Insectcells are transformed by a properly modified method that is describedin, for example, Bio/Technology, vol. 6, 47, (1988), etc. Animal cellsare transformed by a properly modified method that is described in, forexample, Cell Technology suppl.8/New Cell Technology ExperimentalProtocol, 263 (Issued from Shujunsha Co. Ltd. in 1995.) and Virology,vol. 52, 456 (1973), etc.

By culturing the above transformants under the presence of a specificcompound and comparing the measured amounts of the gene product in thepreparation, its capacity to stimulate the promoter activity can beknown. The transformants can be cultured by a method well-known initself.

When the hosts for transformants are yeasts, for example;Berkholder-minimum medium (Proc. Natl. Acad. Sci. USA, vol.77, 4505(1980)) is enumerated as a culture medium. The medium is suitablyadjusted to approx pH 5 to 8. It is usually cultured for approx 24 to 72hours at approx 20 to 30° C., and is ventilated and is stirred, ifnecessary.

When the hosts for transformants are insect cells, the additive such as10% inactivated bovine serum, etc. is properly added to Grace's InsectMedium (Nature, vol. 195, 788 (1962)). The medium is suitably adjustedto approx pH 6.2 to 6.4. It is usually cultured for approx 3 to 5 daysat approx 27° C. and is ventilated and is stirred, if necessary.

When the hosts for transformants are animal cells, for example,Dulbecco's modified Eagle medium (Nacalai Tesque, Inc.) containingapprox 5-20% of the fetal bovine serum is used as a culture medium. Themedium is suitably adjusted to approx pH 6 to 8. It is usually culturedfor approx 15 to 60 hours at approx 30 to 40° C. and is ventilated andis stirred, if necessary.

Because the DNA of the present invention is a nucleotide fragmentincluding the promoter region that contains either of the regulatorysequence PPRE or LRH-RE in human adiponectin promoter region, or both, acompound or salt thereof that enhances the human adiponectin promoteractivity can be screened by using the transformants. As follows, itconcretely explains the screening method, the screening kit, and thecompound or salt thereof that enhances the promoter activity of humanadiponectin gene, which is obtained with the screening method and thescreening kit.

(1) The Method Screening the Compound or Salt Thereof That Enhances thePromoter Activity of Human Adiponectin Gene

The transformants transformed with the DNA including the promoter regionthat contains either of the regulatory sequence PPRE or LRH-RE in humanadiponectin promoter region or both, are usefull for screening thecompound or salt thereof that enhances the promoter activity of humanadiponectin gene.

A method to determine whether the compound or salt thereof can enhancethe promoter activity of human adiponectin gene is characterized bycomparing the amount of polypeptide expression using transformantscontaining the regulatory sequence of the present invention, with thatusing transformants without the regulatory sequence.

The subject compounds include peptides, proteins, non-peptide compounds,synthetic compounds, and fermentation products, etc. The compounds maybe new compounds and well-known compounds. The polypeptides coded by theabove structural genes (suitable reporter genes), etc. are used as theexpressed polypeptides. The measuring method of the polypeptideexpression includes, for example, measuring the luciferase activity by amethod according to Brasier et al.'s method (Biotechniques, vol. 7,1116-1122 (1989)).

(2) The Kit for Screening the Compound or Salt Thereof That Enhances thePromoter Activity of Human Adiponectin Gene

The kit of the present invention for screening the compound or saltthereof that enhances the promoter activity of human adiponectin gene ischaracterized as using the above transformants, and the following areenumerated as the example.

A Cell Culture Medium:

-   -   Dulbecco's modified Eagle medium (Nacalai Tesque, Inc.)        containing 5% of inactivated fetal bovine serum (JRH        Biosciences, Inc.)

A plasmid for measuring the human adiponectin promoter activity: the oneof which the DNA containing the regulatory sequence PPRE and LRH-RE inhuman adiponectin promoter region of the present invention is insertedin multi-cloning site of pGL3-basic vector (Promega corporation)containing a luciferase gene.

A plasmid for nuclear receptor expression: Each full-length cDNA ofhuman PPARγ, human RXRα, and human LRH-1 is obtained by PCR using thehuman cDNA library (Clontech) as a template. The one of which theobtained cDNA and the expression plasmid for mammalian cells are cutwith the same restriction enzymes and connected is used.

Host Cell Lines:

HEK293 cells (human fetal kidney-derived cell lines, obtained fromATCC).

Subject compounds: Before using, the aqueous solution stored at 4° C. or−20° C. is diluted to 50 μM with cell culture medium. Water-insolublecompounds are dissolved with dimethyl sulphoxide and ethanol, etc.

(3) Screening Method

HEK293 cells are seeded into 96-well multiplate (Nalge NuncInternational) and are incubated under 5% of CO₂, at 37° C., forovernight. Transfection is carried out according to a method describedin Lu et al.'s report (Mol. Cell, 6:507-515, 2000). 50 ng/well of theplasmid for the measure of promoter activity in human adiponectinpromoter of the present invention and 15 ng/well of the plasmidexpressing the nuclear receptor are transiently transfected by calciumphosphate method. 8 hours after transfection, subject compound diluentof one-fifth amount of culture supernatant is added. The culture isincubated under 5% of CO₂, at 37° C., for 18 hours and then 100 μl/wellof Pickagene LT reagent (TOYO INK. CO., LTD.) is added. After stirringfor 5 minutes, the luminescence intensity is measured by Lmax microplateluminometer (Molecular Devices corporation).

If a subject compound explored by using the above screening method orthe screening kit enhances the promoter activity of human adiponectingene, it can increase the production and secretion of adiponectin inadipose tissue and thereby increase the plasma adiponectinconcentration. Therefore, the compound can be used as a preventiveand/or therapeutic medicine for metabolic disorder such as diabetes,obesity, hypercholesterolemia, hyperlipoproteinemia, hyperlipidemia,arteriosclerosis, hypertonia, circulatory system diseases, andhyperphagia, etc.

In addition, the compound can be used as a preventive and/or therapeuticmedicine for various syndromes (Syndrome X, metabolic syndrome, multiplerisk factor syndrome, insulin resistance syndrome, deadly quartet,visceral fat syndrome, etc.) of which the above disease are an etiology.

Non-toxic salts of the compound obtained by the above screening methodor the screening kit include, for example, alkali metal salts(potassium, sodium, lithium, etc.), alkaline earth metal salts(calcium,magnesium, etc.), ammonium salts (tetramethylammonium,tetrabutylammmonium, etc.), organic amine salts (triethylamine,methylamine, dimethylamine, cyclopentylamine, benzylamine,phenethylamine, piperidine, monoethanolamine, diethanolamine,tris(hydroxymethyl)methylamine, lysine, arginine, N-methyl-D-glucamine,etc.), acid-addition salts (inorganic acid salts (hydrochloride,hydrobromate, hydroiodate, sulfate, phosphate, and nitrate, etc.), andorganic acid salts (acetate, trifluoroacetate, lactate, tartrate,oxalate, fumarate, maleate, benzoate, citrate, methane sulfonate, ethanesulfonate, benzene sulfonate, toluene sulfonate, isethionate,glucuronate, gluconate, etc.), etc.

Non-toxic salts of the compound in the present invention also includesolvates thereof, or solvates of alkali (earth) metal salts, ammoniumsalts, organic amine salts, and acid-addition salts of the abovecompound in the present invention.

The solvates are preferably non-toxic and water-soluble. Appropriatesolvates, for example, solvates such as water, alcohol solvents(ethanol, etc.), etc. are included.

When the compound of the present invention or salt thereof is used asthe preventive or therapeutic medicine, it is used as a solidpreparation and a liquid medicine for oral administration, andinjections, external preparations, and suppositoriums, etc. forparenteral administration.

The solid composition for oral administration includes a tablet, a pill,a capsule, a dispersing powder, a granule, etc. The capsule includes ahard capsule and a soft capsule.

In such solid composition, an active compound is used by beingformulated according to usual methods as it is, or by being mixed withinert diluents (lactose, mannitol, glucose, hydroxypropyl cellulose,microcrystalline cellulose, starch, etc.), binders(hydroxypropylcellulose, polyvinyl pyrrolidone, and magnesiumaluminometasilicate, etc.), disintegrators (calciumcarboxymethylcellulose, etc.), lubricants (magnesium stearate etc.),stabilizers, and solubilizers (glutamate and aspartic acid, etc.). Itmay be coated with coating agents (sugar, gelatin, hydroxypropylcellulose or hydroxypropyl cellulose phthalate, etc.) or be coated withtwo or more films, if necessary. Furthermore, it includes a capsulecomprising of absorbable material such as gelatin.

The liquid composition for oral administration includes pharmaceuticallyacceptable emulsions, solutions, syrups and elixirs. In such liquidcomposition, an active compound is dissolved, suspended, and emulsifiedin general inert diluents (purified water, ethanol, and mixture thereof,etc.). Furthermore, this liquid composition may also contain wettingagents, suspending agents, emulsifying agents, sweetening agents,flavoring agents, perfuming agents, preserving agents, and bufferagents, etc.

The injection for parenteral administration includes solutions,suspensions, emulsions, and solid injections that are dissolved orsuspended in a solvent before using. The injection is used bydissolving, suspending, or emulsifying an activator into a solvent. Forexample, distilled water for injection, saline, vegetable oil, alcoholssuch as propylene glycol, polyethyleneglycol, and ethanol, and thecombination thereof is used as a solvent. Furthermore, this injectionmay include stabilizers, solubilizers (glutamic acid, aspartic acid,POLYSORBATE 80®, suspending agent, emulsifying agents, soothing agents,buffer agents, and preservatives, etc. These are manufactured by beingsterilized or the aseptic manipulation in the final process. The sterilesolid preparation can be used by being manufactured as a freeze-driedpreparation, and being sterilized or dissolved in distilled water forinjection or other solvent before using.

The external preparation for parenteral administration includes, forexample, ointments, gels, creams, fomentations, patches, embrocations,aerosols, inhalants, sprays, aerosols, eye drops, and nasal drops, etc.These include an activator and are manufactured by the well-known methodor the formula usually used.

The ointment is manufactured by the well-known method or the formulausually used. For example, it is manufactured by mixing and melting theactivator into a basis. The ointment base is chosen from the well-knownone or the one usually used. For example, they are used alone or bybeing mixed with two or more kinds chosen from higher fatty acid, higherfatty acid ester (adipic acid, myristic acid, palmitic acid, stearicacid, oleic acid, adipic acid ester, myristic acid ester, palmitate,stearic acid ester, and oleic acid ester, etc.), waxs (yellow wax,spermaceti, and ceresin, etc.), surfactants (polyoxyethylene alkyl etherphosphate etc.), higher alcohols (cetanol, stearyl alcohol, andcetostearyl alcohol, etc.), silicone oils (dimethylpolysiloxane etc.),hydrocarbons (hydrophilic petrolatum, white petrolatum, purifiedlanolin, and liquid paraffin, etc.), glycols (ethylene glycol,diethylene glycol, propylene glycol, polyethylene glycol, and macrogol,etc.), vegetable oils (castor oil, olive oil, sesame oil, and oil ofturpentine, etc.), animal oils (mink oil, yolk oil, squalane, andsqualene, etc.), water, absorption enhancer, and poisoned inhibitor.Further, they may include moisturizing agents, preservatives,stabilizing agents, anti-oxidants, and flavors, etc.

The gel is manufactured by the well-known method or the formula usuallyused. For example, it is manufactured by melting an activator into abasis. The gel base is chosen from the well-known one or the one usuallyused. For example, it is used alone or by being mixed with two or morekinds chosen from lower alcohols (ethanol and isopropyl alcohol, etc.),gelatinizers (carboxymethylcellulose, hydroxyethyl cellulose,hydroxypropylcellulose, and ethyl cellulose, etc.), neutralizers(triethanolamine and diisopropanolamine, etc.), surfactants(mono-stearic acid polyethylene glycol, etc.), gums, water, absorptionenhancers, and poisoned inhibitors. Further, it is includepreservatives, anti-oxidants, and flavors, etc.

The cream is manufactured by the well-known method or the formulausually used. For example, it is manufactured by melting or emulsifyingan activator into a basis. The cream base is chosen from the well-knownone or the one usually used. For example, they are used alone or bybeing mixed with two or more kinds chosen from higher fatty acid esters,lower alcohols, hydrocarbons, polyhydric alcohols (propylene glycol,1,3-butylene glycol, etc.), higher alcohols (2-hexyl decanol andcetanol, etc.), and emulsifiers (polyoxyethylene alkyl ethers and fattyacid esters, etc.), water, absorption enhancers, and poisonedinhibitors. Further, it may include preservatives, anti-oxidants, andflavors, etc.

The fomentation is manufactured by the well-known method or the formulausually used. For example, it is manufactured by melting an activatorinto a basis and spreading it and rolling on the support after kneading.The fomentation base is chosen from the well-known one or the oneusually used. For example, they are used alone or by being mixed withtwo or more kinds chosen from thickeners (polyacrylic acid, polyvinylpyrrolidone, arabic gum, starch, gelatin, and methyl cellulose, etc.),humectants (urea, glycerin, and propylene glycol, etc.), and fillers(china clay, flower of zinc, talc, calcium, and magnesium, etc.), water,absorption enhancers, and poisoned inhibitors. Further, it may includepreservatives, anti-oxidants, and flavors, etc.

The patch is manufactured by the well-known method or the formulausually used. For example, it is manufactured by melting an activatorinto a basis and spreading it and rolling on the support after kneading.The patch base is chosen from the well-known one or the one usuallyused. For example, they are used alone or by being mixed with two ormore kinds chosen from high molecular basis, oils, fats, higher fattyacids, tackifiers, and poisoned inhibitors. Further, it may includepreservatives, anti-oxidants, and flavors, etc.

The liniment is manufactured by the well-known method or the formulausually used. For example, it is manufactured by dissolving, suspending,or emulsifying an activator into alone, or two or more kinds chosen fromwater, alcohols (ethanol and polyethylene glycol, etc.), higher fattyacids, glycerins, soaps, emulsifiers, suspending agents, etc. Further,it may include preservatives, anti-oxidants, and flavors, etc.

The aerosol, the inhalant, and the spray may contain stabilizers such assodium hydrogen sulfite, buffers giving isotonicity, and isotonic agentssuch as, for example, sodium chloride, sodium citrate, and citratesbesides the general diluent. Production methods of the spray have beendescribed in U.S. Pat. Nos. 2,868,691 and 3,095,355.

Usually, the nasal drop is quantitatively spray-administered into thenasal cavity as a solution and a powder containing a medicine or byusing the dedicated nasal drip machine or the sprayer.

The eye drop for parenteral administration includes eye drops, eyesuspensions, eye emulsions, dissolution type of eye solutions beforeusing, and eye ointment.

For example, they are used by dissolving, suspending, and emulsifying anactivator into a basis. As a solvent of eye drop, for example, sterilepurified water, saline, other water solvent, or injectable nonaqueoussolvent (for example, vegetable oil etc.), and combination thereof areused.

For example, the ophthalmic solutions are made with tonicity agents(sodium chloride and concentrated glycerin, etc.), buffers (sodiumphosphate and sodium acetate, etc.), surfactants (polysorbate 80 (tradename), polyoxyl 40 stearate, and polyoxyethylene hydrogenated castoroil, etc.), stabilizers (sodium citrate and disodium edetate, etc.), andpreservatives (benzalkonium chloride and paraben, etc.), etc., which areproperly selected, if necessary. These are sterilized in the finalprocess or manufactured by the aseptic manipulation. The manufacturedsterile solid preparation, for example, freeze-drying product can beused by being sterilized or being dissolved to sterile purified water orother sterile solvent before using.

Inhalants for parenteral administration may include aerosol agents,inhalant powders or inhalant liquids, which may be used by beingdissolved or suspended in water or other suitable media before using.These inhalants are manufactured according on a well-known method. Forexample, inhalant liquids are prepared by being properly selected frompreservatives(benzalkonium chloride and paraben, etc.), coloring agents,buffers (sodium phosphate and sodium acetate, etc.), tonicity agents(sodium chloride and concentrated glycerin, etc.), thickeners(carboxyvinyl polymer, etc.), and absorption enhancers, etc., ifnecessary.

Inhalant powders are prepared by being properly selected from lubricants(stearic acid and the salt, etc.), binders (starch and dextrin, etc.),fillers (lactose and cellulose, etc.), coloring agents, preservatives(benzalkonium chloride and paraben, etc.), and absorption enhancers,etc., if necessary.

When the inhalant liquid is administered, a sprayer (atomizer andnebulizer) is usually used, and when the inhalant powder isadministered, an inhalation administering machine for powder is usuallyused.

As other compositions for parenteral administration, suppositories forintrarectal administration, and pessaries for administering in vagina,which contain activators and are prescribed with common procedure, areincluded.

Because the obtained preparations in this way are safe and low-toxic,they can be administered to, for example, human and mammals (forexample, rat, mouse, rabbit, cat, dog, and monkey, etc.). The dosage isdetermined depending on age, body weight, symptom, therapeutic effect,administration route, duration of the treatment and the like. Generally,1 ng to 100 mg per an adult is orally administered once to several timesper a day, or 0.1 ng to 10 mg per an adult is parenterally administered(preferably, nosedrop, ophthalmic solution, ointment) once to severaltimes per a day, or intravenously administered for 1 to 24 hours per aday, continuously.

Since the dose changes depending on various conditions as describedabove, there are also cases in which doses lower or greater than theabove dose may be used.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the DNA sequence containing human adiponectin promoterregion;

FIG. 2 shows the structure of human adiponectin promoter/reporterplasmid. DNA and promoter-deletion constructs;

FIG. 3 shows transcriptional activities of human adiponectinpromoter/reporter plasmid DNA enhanced by constitutively-active PPARγand RXRα;

FIG. 4 shows transcriptional activities of human adiponectinpromoter/reporter plasmid DNAs and promoter-deletion constructs;

FIG. 5 shows comparison of PPRE nucleotide sequences of the genes thatare transcriptionally regulated by PPARγ/RXR heterodimer, where thehuman adiponectin PPRE sequence is SEQ ID NO:16, where the mouse aP2PPRE sequences are SEQ ID NO:17 (upper) and SEQ ID NO:18 (lower), wherethe mouse c-Cb1 binding protein PPRE sequence is SEQ ID NO:19, where themouse LXRαPPRE sequence is SEQ ID NO:20, and where the mouse aquaporinadipose PPRE sequence is SEQ ID NO:21;

FIG. 6 shows PPRE sequence (SEQ ID NO:22) in human adiponectin gene anda structure of human adiponectin promoter/reporter plasmid DNA mutatedin PPRE sequence;

FIG. 7 shows transcriptional activities of the mutated promoter/reporterplasmid DNAs by constitutively-active PPARγ and RXRα;

FIG. 8 shows direct binding (gel shift assay) of PPARγ/RXR heterodimerto the regulatory sequence PPRE of human adiponectin promoter;

FIG. 9 shows comparison of LRH-RE nucleotide sequences contained in thegenes that are transcriptionally regulated by LRH-1;

FIG. 10 shows LRH-RE sequence in human adiponectin gene and a structureof human adiponectin promoter/reporter plasmid DNA mutated in LRH-REsequence;

FIG. 11 shows transcriptional activities of human adiponectinpromoter/reporter plasmid DNAs enhanced by pioglitazone when PPARγ/RXRheterodimer and/or LRH-1 is/are expressed;

FIG. 12 shows direct binding (gel shift assay) of LRH-1 to theregulatory sequence LRH-RE of human adiponectin promoter; and

FIG. 13 shows transcriptional activities of the wild-type or mutatedhuman adiponectin promoter/reporter plasmid DNAs enhanced bypioglitazone in differentiated adipocytes.

BEST MODE FOR CARRYING OUT THE INVENTION

The following examples illustrate the present invention, but do notlimit the present invention.

EXAMPLE 1 Construction of Human Adiponectin Promoter/Reporter PlasmidDNA

A DNA containing human adiponectin promoter region was amplified by PCRmethod using DNA fragments obtained according to Takahashi et al.'smethod as a template (Int. J. Obes. Relat. Metab. Disord., 24, 861-868(2000)). Concretely, a clone containing human adiponectin gene wasobtained from human P1-derived artificial chromosome (PAC) DNA pool(GENOMESYSTEMS, INC.), and was digested by restriction enzymes BamHI andXbaI. 2.9 kb of the DNA fragments among the obtained fragments containedthe 5′-upstream region of human adiponectin gene (Int. J. Obes. Relat.Metab. Disord., 24, 861-868 (2000)). Using the DNA fragments as atemplate and primers (5′-TTT CGG GGT ACC GCT TCT AGG CCA GAG CTG GGTTC-3′(SEQ ID NO:5) and 5′-TTT CGG GAG CTC CTG CAG TCA GAA TGG AAG TGAGAA-3′ (SEQ ID NO:6)), the DNA fragments containing human adiponectinpromoter region were amplified, and the nucleotide sequence wasdetermined. FIG. 1 shows the nucleotide sequence. The amplified DNAfragments and pGL3 basic plasmid (Promega Corporation) having fireflyluciferase gene were digested by restriction enzymes KpnI and SacI, andboth were ligated. FIG. 2 shows the schematic structure. Hereafter, thishuman adiponectin promoter/reporter plasmid is abbreviated asp(−908)/LUC.

EXAMPLE 2 Assay of Transcriptional Activities of Human AdiponectinPromoter/Reporter Plasmid DNAs

Luciferase assay was performed using p(−908)/LUC constructed asdescribed in Example 1 and constitutively-active PPARγ/RXR heterodimer.By transfection of expression plasmid encoding each chimera nuclearreceptor (they are abbreviated as VP16-PPARγ and VP16-RXRα.) of which atranscriptional active region of herpes virus protein VP16 has beenlinked to nuclear receptor PPARγ or RXRα, the promoter activity wasassayed. These chimera nuclear receptors are active the transcription ofa responsive gene even in the absence of each ligand (Mol. Endocrinol.,16, 1040-1048 (2002)). As a reporter plasmid, p(−908)/LUC constructed inExample 1 was introduced together with the nuclear receptor expressionplasmids. HEK293 cells derived from human fetus kidney were used as ahost cell. The cells were seeded into 96-well:multiplate (Nalge NuncInternational) and were cultured in Dulbecco's modified Eagle medium(Nacalai Tesque, Inc.) containing 5% of inactivated fetal bovine serumunder 5% of CO₂, at 37° C., for overnight, and were transformed.Transfection was performed as described in Lu et al.'s report (Mol.Cell, 6, 507-515 (2000)). 50 ng per well of the reporter plasmidp(−908)/LUC, 20 ng per well of beta-galactosidase expression plasmid,and 15 ng of each nuclear receptor expression plasmid were respectivelytransfected by calcium phosphate method. Luciferase activity andbeta-galactosidase activity as internal standard were respectivelymeasured by using Lmax microplate luminometer and Emax microplate reader(Molecular Devices corporation) 26 hours after transfection. The resultwas presented as a relative activity that divides the luciferaseactivity with the beta-galactosidase activity. FIG. 3 shows the result.In cells expressing both VP16-PPARγ and VP16-RXRα, the remarkableincrease in luciferase activity was seen. When only VP16, VP16-PPARγ, orVP16-RXR was expressed, no change was seen. According to these results,it was thought that PPARγ/RXR heterodimer could act on human adiponectinpromoter.

EXAMPLE 3 Identification of Regulatory Sequences in Human AdiponectinPromoter Region (PPRE)

To identify the region in human adiponectin promoter leading to theincrease in the transcriptional activity observed in Example 2, deletionconstructs of human adiponectin promoter were made as shown at thebottom of FIG. 2. By PCR method using the above p(−908)/LUC as atemplate and primers designed to obtain a target length of the promoter,the deletion promoter fragment was amplified. The amplified DNAfragments and pGL3 basic plasmid were digested by restriction enzymesand connected. By using each deletion construct as a reporter andtransiently expressing both expression plasmids of VP16-PPARγ andVP16-RXRα in HEK293 cells, the luciferase activity was measured aspreviously described in Example 2. FIG. 4 shows the result. By deletinga sequence from −286 bp to −267 bp in human adiponectin promoter, theincrease in luciferase activity by VP16-PPARγ and VP16-RXRα disappeared.As a result, it was presumed that the regulatory sequence PPRE shouldexist between −286 bp and −267 bp in human adiponectin promoter region.

FIG. 5 shows the nucleotide sequence of PPRE in the promoter region ofgenes that has been reported to be transcriptionally controlled byPPARγ/RXR heterodimer (Genes Dev., 8, 1224-1234 (1994), J. Biol Chem.,275, 9131-9135 (2000), Mol. Cell, 7, 161-171 (2001), J. Biol. Chem.,276, 48572-48579 (2001)). All sequences have a similar structurecomprising of 13 bases called “direct repeat 1(DR1)”. By examining thenucleotide sequence from −286 bp to −267 bp in human adiponectinpromoter region in detail, it turned out that a sequence similar to thereported PPRE existed from −273 bp to −285 bp (FIGS. 5 and 6). Toconfirm that the presumed sequence functions as the regulatory sequencePPRE, a 2-bp mutation was introduced into a presumed sequence of thereporter plasmid as shown in FIG. 6. The mutation was introduced usingQuikChange Site-Directed Mutagenesis kit (Stratagene Corporation)according to a supplyer's protocol. To compare the transcriptionalactivity of a PPRE-mutant reporter plasmid with that of a wild-typereporter, the luciferase activity was measured as previously describedin Example 2. FIG. 7 shows the result. Using a mutated reporter plasmid,the increase in luciferase activity disappeared. The result suggestedthat the sequence presumed to be PPRE could function as a regulatorysequence and could be necessary for the transcriptional activation ofhuman adiponectin promoter by PPARγ/RXR heterodimer.

EXAMPLE 4 Direct Binding to the Regulatory Sequence PPRE of PPARγ/RXRHeterodimer

To confirm the direct binding of PPARγ/RXR heterodimer to the regulatorysequence PPRE that had been identified from −285 bp to −273 bp in humanadiponectin promoter region, gel shift assay was performed according tothe method previously described (J. Biol. Chem., 276, 48572-48579(2001)). For the binding reaction, human PPARγ and RXRα protein weresynthesized in vitro by using TNT T7 Quick CoupledTranscription/Translation Systems (Promega corporation) according to asupplyer's protocol. For a labeled probe, oligoDNAs (5′-TGG TTT TGA CTTTTG CCC CAT CTT C-3′ (SEQ ID NO:7) and 5′-GAA GAT GGG GCA AAA GTC AAAACC A-3′ (SEQ ID NO:8)) having a nucleotide sequence from −291 bp to−267 bp in human adiponectin promoter region were labeled using[γ-32P]ATP (Amersham Biosciences K.K.) and T4 polynucleotide kinase(TaKaRa Shuzo Co. Ltd.). An underlined sequence represents PPRE. Thebinding reaction was performed in 20 μl of a solution including 1 μg ofpoly (dI-dC), 1 μl of the in vitro synthesized nuclear receptorsolution, and 200,000 cpm of the labeled probe. After mixing, it wasincubated for 20 minutes at 25° C. and was left for 15 minutes at 4° C.The labeled probe binding to nuclear receptors was separated from thefree probe by electrophoresis using 4% of polyacrylamide gel. Theelectrophoresis was executed using 0.5× TBE buffer (45 mM Tris, 45 mMboric acid, and 1 m M EDTA) for 90 minutes under a voltage of 200V.After the electrophoresis, the gel was dried and analyzed by BAS2500system (Fuji Photo Film Co., Ltd.). To confirm that the labeled probebinds to nuclear receptors specifically, the competitive reactions wereexecuted. The competitive reactions were executed by adding 10 or 50times-higher concentration of the unlabeled probe containing wild-typePPRE or mutated PPRE (5′-TGG TTT TGA CTT TTG ttC CAT CTT C-3′ (SEQ IDNO:9), and 5′-GAA GAT GGa aCA AAA GTC AAA ACC A-3′ (SEQ ID NO:10)). Themutated nucleotides are represented by small letters. An underlinedsequence represents PPRE. FIG. 8 shows the result. The arrow indicates aband of the labeled probe binding to nuclear receptors. When both PPARγand RXRα existed, the band of the complex was detected (lane 4). Whenadding an excessive amount of unlabeled oligoDNAs whose PPRE was intact,the band was disappeared in a concentration dependent manner (lane 5 andlane 6). However, when adding unlabeled oligoDNAs with mutated PPRE, thecompeting effect seen with the wild-type oligoDNAs was attenuated (lane7 and lane 8). These results revealed that PPARγ/RXR heterodimerspecifically binds to the regulatory sequence PPRE identified in humanadiponectin promoter region.

EXAMPLE 5 Identification of the Regulatory Sequence (LRH-RE) in HumanAdiponectin Promoter Region

To clarify whether a regulatory sequences other than PPRE exists inhuman adiponectin promoter region, the nucleotide sequence ofadiponectin promoter was intensively analyzed. As a result, wediscovered a sequence presumed to be the regulatory sequence LRH-RE(LRH-1 responsive element) to which another nuclear receptor calledLRH-1 (Liver Recepter Homologue-1) can bind. FIG. 9 shows the nucleotidesequence of LRH-RE in gene promoter region that has been reported to betranscriptionally regulated by LRH-1 (Mol. Cell, 6, 507-515 (2000),Proc. Natl. Acad. Sci. U.S.A., 96, 6660-6665 (1999), J. Biol. Chem.,276, 24767-24773 (2001), J Biol. Chem., 275, 17793-17799 (2000)). Onlyone base was different between the LRH-RE sequence found in humanadiponectin promoter and the LRH-RE in rat and human CYP7A1 genepromoter. FIG. 10 shows the position of the presumed LRH-RE. Thepresumed LRH-RE was located from −237 bp to −229 bp of the transcriptionstart site of human adiponectin gene. Because the presumed LRH-RE existsin human adiponectin promoter, it was thought that adiponectin promotercould be transcriptionally regulated by nuclear receptor LRH-1. Then,using the above p(−908)/LUC as a reporter plasmid, the expressionplasmids of which PPARγ, RXRα, and LRH-1 can be expressed respectivelywere transiently expressed in HEK293 cells as previously described inExample 2. Pioglitazone, a PPARγ agonist, was added at 1 μM, 8 hoursafter transfection. FIG. 11 shows the result. The luciferase activitywas doubled by the expression of both PPARγ and RXRα. The activity rosefurther in the presence of pioglitazone (lane 2). On the other hand, theincrease in the luciferase activity was not seen with only LRH-1expression (lane 3). However, when PPARγ, RXRα, and LRH-1 were expressedsimultaneously, the luciferase activity was further doubled comparedwith that in expressing both PPARγ and RXRα. The effect was also seen inthe presence of pioglitazone (Lane 4 compared with lane 2). Theseresults revealed that LRH-1, which doesn't act alone, could augment thehuman adiponectin promoter activity stimulated by PPARγ/RNR heterodimer.Then, to confirm that the action of LRH-1 depends on the presumedLRH-RE, a 2-bp mutation was introduced into the presumed LRH-RE of thereporter plasmid as shown in FIG. 10. As shown in FIG. 11, the increaseby PPARγ and RXRα was not influenced by LRH-RE mutation (Lane 6 comparedwith lane 2.). On the other hand, the further increase in promoteractivity by LRH-1 seen with wild-type reporter (lane 4 compared withlane 2.) was completely disappeared (lane 8 compared with lane 6.).These results revealed that the action of LRH-1 would depend on thepresumed LRH-RE in human adiponectin promoter region. And, the presumedLRH-RE was thought to function as a regulatory sequence.

EXAMPLE 6 Direct Binding of LRH-1 to the Regulatory Sequence LRH-RE

To confirm the direct binding of LRH-1 to the regulatory sequence LRH-REfound in human adiponectin promoter region, gel shift assay wasexecuted. Gel shift assay was performed as described in Example 4. LRH-1protein used was synthesized in vitro using human LRH-1 expressionplasmid as template. OligoDNAs (5′-AAT AAG GGT CAA GGC CTG GAA ACA C-3′(SEQ ID NO:11) and 5′-GTG TTT CCA GGC CTT GAC CCT TAT T-3′ (SEQ IDNO:12)) having a nucleotide sequence from −245 bp to −221 bp in humanadiponectin promoter region were used for a labeled probe. An underlinedsequence represents LRH-RE. In the competitive reaction, identicaloligoDNAs to the labeled probe were used as wild-type competitors. Asmutant competitors, oligoDNAs (5′-AAT AAG GGT CAA ccC CTG GAA ACA C-3′(SEQ ID NO:13) and 5′-GTG TTT CCA GGg gTT GAC CCT TAT T-3′. (SEQ IDNO:14)) mutated as described in FIG. 10 were used. 10 times or 50 timeshigher concentration of each unlabeled oligoDNAs was added into thereaction. The small letter means a mutated base. The underlined sequencerepresents LRH-RE. FIG. 12 shows the result. The complex of LRH-1 andthe wild-type labeled probe was detected as pointed by an arrow (lane2). When an excessive amount of wild-type unlabeled oligoDNAs wereadded, the band was disappeared in a concentration dependent manner(lane 3 and lane 4). On the other hand, when an excessive amount ofmutated unlabeled oligoDNAs added, the band of the complex slightlyattenuated (lane 5 and lane 6). These results revealed that LRH-1 couldspecifically bind to the regulatory sequence LRH-RE identified in humanadiponectin promoter region.

EXAMPLE 7 Roles of the Identified Regulatory Sequence PPRE and LRH-RE inAdipocytes

To indicate whether the identified regulatory sequence PPRE and LRH-REcould take part in transcriptional activation of adiponectin gene inadipocytes, the following experiment was performed. Mouse 3T3-L1 cellswere cultured by using 6-well plate (Becton, Dickinson & Company) coatedby type IV collagen and the differentiation was induced in mediumcontaining 5 μg/ml of insulin, 0.5 mM isobutylmethylxanthine, and 1 μMdexamethasone. The 6th-day cells from the differentiation induction wereused for transformation. LipofectAMINE 2000 reagent (InvitrogenCorporation) was used for transfection according to a supplyer'sprotocol. 2 μg of reporter plasmid and 1 μg of beta-galactosidaseexpression plasmid for internal standard per well were used,respectively. The mixture of LipofectAMINE 2000 reagent and plasmidsdiluted with OPTI-MEM were added to cells, which were incubated for 3.5hours. Then, the equivalent volume of medium containing 20% of fetalbovine serum was added to the culture. After 44 hours, the luciferaseactivity and the beta-galactosidase activity were measured. When themedium containing 20% of fetal bovine serum was added, pioglitazone wasadded at 1 μM. FIG. 13 shows the result. When using wild-typep(−908)/LUC reporter, the luciferase activity was 19-times higher thanthat of promoter-less pGL3 basic plasmid. In the presence of 1 μMpioglitazone, further increase was seen. It was 9-times higher thanwithout pioglitazone. The luciferase activity of the PPRE-mutatedpromoter (FIG. 6) remarkably decreased compared with that of thewild-type promoter, and the effect of pioglitazone was not observed atall. When the LRH-RE-mutated promoter (FIG. 10) was used, the luciferaseactivity also decreased compared with that of the wild-type promoter.However, the luciferase activity of the cells processed by pioglitazonehas increased 7.5 times-higher than that without pioglitazone and theresponse to pioglitazone was remained. These results revealed that theidentified regulatory sequence PPRE and LRH-RE played an important rolein the activation of adiponectin promoter in adipocytes. Concurrently,it was thought that these regulatory sequences could deeply take part inadiponectin gene expression in a physiological condition.

INDUSTRIAL APPLICABILITY

The nucleotide sequence PPRE (located between 5′ upstream 273rd and285th of the transcription start site in human adiponectin gene) andLRH-RE (located between 5′ upstream 229th and 237th as well as the abovePPRE) are regulatory sequences in human adiponectin promoter region, andare sequences that nuclear receptor PPARγ, RXR, and LRH-1 bind directly,and play important roles in physiological activation of adiponectinpromoter in adipocytes. Transformants transformed with reporter plasmidscontaining the promoter region with these regulatory sequences andsuitable reporter genes approximate the physiological expression patternof human adiponectin gene, and are extremely useful to screentherapeutic drugs for human diseases.

1. A screening method for identifying a compound, or a salt thereof,which enhances human adiponectin promoter activity, comprising the stepsof: (1) transforming a first and second cell with (a) a DNA moleculecomprising the nucleotide sequence of SEQ ID NO:1 operatively linked toa DNA sequence encoding a reporter, (b) an expression plasmid encoding ahuman PPARγ protein and (c) an expression plasmid encoding a human RXRαprotein; (2) contacting said first cell with a diluent containing a testcompound; (3) contacting said second cell with a diluent lacking saidtest compound; (4) comparing the amount of said reporter that isproduced by said first cell and said second cell, and wherein when agreater amount of said reporter is produced by said first cell aftercontacting with said diluent containing said test compound compared tothe amount of said reporter produced by said second cell aftercontacting with said diluent lacking said test compound, said testcompound is identified as enhancing human adiponectin promoter activity.2. The method according to claim 1, wherein said test compound regulateshuman PPARγ activity.
 3. The method according to claim 1, wherein saidtest compound regulates human RXRα activity.
 4. The method according toclaim 1, wherein said test compound regulates human LRH-1 activity. 5.The screening method according to claim 1, wherein step (1) furthercomprises transforming said first and second cell with an expressionplasmid encoding a human LRH-1protein.